Picornaviruses replicate via RNA intermediates in the cytoplasm of infected cells, producing dsRNAs that activate antiviral host responses, including the OAS-RNase L pathway. Recently, we discovered a viral RNA molecule that inhibits the endoribonuclease domain of RNase L, and we characterized individual steps of RNA replication, including reiterative transcription mechanisms required for viral RNA polyadenylation. Enterovirus RNA polymerase (3Dpol) is well characterized at the structural level, yet, it is still unclear how conserved amino acid sequences and structures in the polymerase mediate distinct aspects of viral RNA replication. We intend to focus on a dsRNA clamp of 3Dpol that holds RNA products of viral replication as they exit the polymerase. Our preliminary data indicate that mutations in the dsRNA clamp of 3Dpol are maintained in infectious virus; however, these mutations impact the polyadenylation of viral RNA, the fidelity of viral RNA replication, the sensitivity of virus to ribavirin and virus fitness. We expect these mutations will also affect virl RNA recombination. After completing the experiments described in this application, we will better understand the structural features of 3Dpol and the manner in which they function. These studies are important for our understanding of antiviral drugs that target viral polymerases and the mechanisms by which recombinant viruses arise in nature, including recombination among wildtype enteroviruses and poliovirus vaccines. Ribonuclease L (RNase L) is an endoribonuclease associated with antiviral and antibacterial defense, cancer and lifespan. Despite the biological significance of RNase L, the RNAs cleaved by this enzyme are poorly defined. We plan to use novel deep sequencing methods to identify the host and viral RNAs targeted by RNase L and determine the functional effect of cleavage in host and viral RNAs. After completing the experiments described in this application, we will better understand how RNase L exerts its antiviral activity and how viruses overcome the antiviral activity of RNase L. Our study will also reveal how RNase L-dependent cleavage sites in 18S rRNA impact ribosome function; thereby revealing how one molecular target of RNase L impacts multiple phenotypes (antiviral activity, apoptosis, cancer and lifespan). New deep sequencing methods provide unprecedented opportunities to better understand virus replication and pathogenesis. During the previous funding period, we developed and validated cyclic phosphate cDNA synthesis and Illumina sequencing methods which reveal the frequency and location of antiviral endoribonuclease cleavage sites in host and viral RNAs. Other members of the research community developed circle cDNA sequencing to measure the fidelity of viral RNA replication. Together, these two deep sequencing methods provide the means for substantial new insights into the host-virus interactions that determine the outcomes of virus infections. We intend to use these new deep sequencing methods, in conjunction with other well-established experimental conditions, to build upon recent discoveries.